Hedgehog Signaling Protocols

The second edition of this volume provides novel protocols for the study of newly discovered function and modifications of components of the Hh pathways. The first few chapters discuss methods for stable delivery of soluble Hh proteins in cell cultures that can be adapted for in vivo studies, and mathematical models for studying Hh proteins gradient formation in tissues. The next few chapters explore studies in cell culture using fibroblasts that respond readily to Hh proteins. The book goes on to present protocols for evaluation of transcriptional activity of the Gli transcription factors in cell cultures and determine the expression level of endogenous Gli proteins. The final chapters talk about metabolic effects of noncanonical Hh signaling. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and authoritative, Hedgehog Signaling Protocols, Second Edition presents researchers with a comprehensive understanding of the Hh pathway and its function in development and diseases.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Contenu

Controlled Delivery of Sonic Hedgehog with a Heparin-Based Coacervate.- In Vivo Imaging of Hedgehog Transport in Drosophila Epithelia.- Modeling Hedgehog Signaling Through Flux-Saturated Mechanism.- Evaluating the Activity of Smoothened Toward G Proteins Using [ 35 S] Guanosine 5'-(3- O -thio)triphosphate ([ 35 S]GTPS).- Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila.- Investigation of Protein-Protein Interactions and Conformational Changes in Hedgehog Signaling Pathway by FRET.- Luciferase Reporter Assays to Study Transcriptional Activity of HH Signaling in Normal and Cancer Cells.- Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR.- Quantitative Immunoblotting of Endogenous Hedgehog Pathway Components.- Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry.- Rapid Screening of Gli2/3 Mutants Using the Flp-In System.- Insights into Gli Factors Ubiquitylation Methods.- Determination of Acetylation of the Gli Transcription Factors.- Efficient Detection of Indian Hedgehog During Endochondral Ossification by Whole Mount Immunofluorescence.- Methods for Detection of Ptc1-Driven LacZ Expression in Adult Mouse Skin.- Determination and Analysis of Cellular Metabolic Changes by Non-Canonical Hedgehog Signaling.